Single crystal EPR studies of the oxidized active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

Abstract

The Ni-A and the Ni-B forms of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F have been studied in single crystals by continuous wave and pulsed EPR spectroscopy at different temperatures (280 K, 80 K, and 10 K). For the first time, the orientation of the g-tensor axes with respect to the recently published atomic structure of the active site at 1.8 A resolution was elucidated for Ni-A and Ni-B. The determined g-tensors have a similar orientation. The configuration of the electronic ground state is proposed to be Ni(III) 3d1/z 2 for Ni-A and Ni-B. The gz principal axis is close to the Ni-S(Cys549) direction; the gx and the gy axes are approximately along the Ni-S(Cys546) and Ni-S(Cys81) bonds, respectively. It is proposed that the difference between the Ni-A and Ni-B states lies in a protonation of the bridging ligand between the Ni and the Fe.

The Synthesis and Electronic Structure of a Novel [Ni‘S4’Fe2(CO)6] Radical Cluster: Implications for the Active Site of the [NiFe] Hydrogenases

“…The relatively low S character (ca. 3±4 %) derived from the bridging thiolate S donors in the SOMO of 1 À contrasts with the electronic structures of the Ni-A, [20,22] -B, [20,22] -C [18,21,22] and -L [18,21,22] forms of the [NiFe] hydrogenases derived from single-crystal EPR studies supported by DFT calculations. These studies reveal the majority of the spin density is oriented along the NiÀ S(Cys533) bond and only a small amount is at the low-spin formally Fe II centre in the Ni-A, Ni-B and Ni-C forms of the enzyme.…”

Relativistic DFT calculation of the reaction cycle intermediates of [NiFe] hydrogenase: a contribution to understanding the enzymatic mechanism

“…The smallest experimental g value is close to the free electron value g e , indicating a d z 2 ground state. In the crystal studies, the g z -axis is found to be approximately oriented along the Ni-SCys533 bond [51]. This is also the direction of the empty free coordination position at the Ni (Fig.…”

“…Additionally, we also compared the calculated g tensor orientation with that obtained from angular-dependent EPR experiments on protein single crystals [50,51]. The smallest experimental g value is close to the free electron value g e , indicating a d z 2 ground state.…”

“…In Table 1, the optimized Ni-Fe distance and g tensor values are presented. Also included are the respective values from X-ray structure analysis [4] and EPR experiments on single crystals [51].…”

“…These are outlined in Table 1. From the analysis of the single crystal EPR spectra of the oxidized states a formal Ni(III) oxidation state has been deduced with the single unpaired electron in a d z 2 orbital [50,51] whereas the iron atom is kept in a formal Fe(II) low spin state due to the strong crystal field imposed by the inorganic ligands CN À and CO. We have therefore adopted a Ni(III)-X-Fe(II) model for all calculations of the oxidized states. Several variations of the bridging ligand 'X' as well as a vacant bridging position were investigated.…”